| Objective:HPV16 opportunistically infects keratin-forming cells in the basal layer of the skin or mucosa,and its gene entry into the nucleus and integration with host genes is an important factor contributing to cellular malignancy.The aim of this study was to observe the differences in expression of HPV16 E6 gene and protein in HPV16 E6recombinant plasmid-transfected human cutaneous squamous cell carcinoma A431 cells,empty plasmid-transfected human cutaneous squamous cell carcinoma A431 cells and blank control group.Methods:Part I:The lentiviral plasmid LV-OE-HPV16 E6 was constructed,and after sequencing to verify the correct sequence,the plasmid was packaged into a high-titer lentivirus;Part II:(1)Cultivation of human cutaneous squamous cell carcinoma A431 cells,observation of cell growth status,determination of the exponential growth period and plotting of cell growth curves;(2)Different concentrations of puromycin solution(0.5,1,1.5,2,2.5μg/m L)were used to design CCK-8 assay to determine the optimal concentration for puromycin resistance screening and to screen human cutaneous squamous cell carcinoma A431 cells successfully transfected with the virus;(3)m RNA and protein expression of HPV16 E6 in the three groups of cells were detected by Real Time quantitative PCR and Western Blotting,respectively.Results:(1)The LV-OE-HPV16 E6 recombinant plasmid was successfully constructed and packaged as a lentivirus with a titer of 2×10~9/m L.(2)The m RNA level and protein expression of human cutaneous squamous cell carcinoma A431 cells transfected with HPV16 E6 recombinant plasmid were significantly higher than those of blank control and human cutaneous squamous cell carcinoma A431 cells with empty plasmid(P<0.05).Conclusion:LV-OE-HPV16 E6 lentivirus successfully infected human cutaneous squamous cell carcinoma A431 cells,and the HPV16 E6 gene and protein were stably expressed in the cells. |
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